Regulation of a large number of genes interacting in pathways and networks underlie the complex properties of tumor cells. Studying these pathways and networks in mammalian cells is limited by the general lack of specific mutants. A gene-specific silencing process has just been discovered in a large number of organisms, RNA interference (RNAi). In these organisms, double stranded (ds) RNA is converted into a sequence-specific intermediate which targets the corresponding mRNA for degradation. Recent evidence suggests that the intermediates are small RNAs of 21-23 nt in length. RNAi has been observed following injection of dsRNA in mouse embryos but has not been reported in somatic mammalian cells in culture. We will further characterize RNAi in Drosophila cells in culture, where it is very efficient. These studies will guide the search for RNAi in mammalian cells in culture. An important goal will be to develop methods using dsRNA or synthetic oligoribonucleotides (and modifications thereof) to specifically silence gene expression in mammalian cells in culture and to use this methodology to study pathways and networks of gene regulation, such as the Rb/E2F pathway. Many fundamental aspects of regulation of transcription are not well understood. Preferential transcription of the immunoglobulin promoter in B cells is mediated by the presence of a conserved octamer sequence which is recognized by the Oct- 1 and Oct-2 proteins. The possibility that the ubiquitously expressed Oct-1 protein is responsible for recognition of the octamer site in B cells is being tested by several experiments including generation of ES cells that are homozygotically mutant for Oct-1. The potential of these ES cells to generate B cells will be determined. In addition, screens of retroviral libraries of cDNAs from B cells are being tested for their potential to convert cells to preferentially transcribing the immunoglobulin promoters. The functions of Oct-2 and OCA-B/Bob-1/OBF-1 proteins in the activities of the heavy chain immunoglobulin 3' enhancer during B-cell activation and in plasma cells will be studied.